During FY16 we accomplished the following; 1. Completed analysis of 3D-organization of the IgH VH domain. Briefly, the 2.5Mb VH domain is folded into CTCF-dependent domains that range in size from 200-400kb. This pre-folded structure is further compacted in a Pax5-dependent manner into two 1Mb-sized domains. These Pax5-dependent domains do not form in the absence of CTCF, whereas Pax5 is not required for formation of CTCF-dependent domains. We interpret these observations to indicate a structural hierarchy for establishing the VH domain. 2. We demonstrated that altered looping of E to VH81X on IGCR1-deleted IgH alleles led to the formation of a new recombination center within the proximal VH genes. We propose that breakdown of recombination order on IGCR1-deleted alleles is the consequence of increased RAG1/2 density at VH81X and its spatial proximity to the DH-JH region of the IgH locus. 3. We generated cell lines that selectively delete specific CTCF binding sites within the proximal VH genes in the context of WT or IGCR1-deleted IgH alleles. Epigenetic and recombination studies are in progress.